Prolonged residence in the phagosomal compartment of host macrophages is critical to the ability of the Brucella spp. to produce disease. Within this environment, the brucellae must resist iron deprivation. B. abortus 2308 can utilize heme as an iron source in vitro. Due to the central role of macrophages in the degradation of erythrocytes and the scavenging of hemoglobin and heme released from damaged cells, heme may represent an important iron source for the brucellae during their intracellular residence in these host phagocytes. Two genetic loci (designated bhuA and bhuTUV) whose products are predicted to be involved in the utilization of heme as an iron source have been identified in B. abortus 2308 and preliminary characterization of a B. abortus bhuA mutant suggests that this heme transporter plays a critical role in virulence in mice. Consequently, the specific aims of the studies outlined in this application are a) to confirm that the products of the genes that we have designated as bhuT, U and Vwork together with BhuA to form a functional heme transporter in 6. abortus 2308;b) to determine the relative contributions of BhuA and BhuTUV to the virulence of 6. abortus 2308 in the mouse model;and c) to define the nature of the iron- and heme-responsive regulation of bhuA and bhuTUV in the B. abortus 2308. The proposed studies should increase our basic understanding of the mechanisms employed by the Brucella spp. and other intracellular pathogens to meet their physiologic need for iron in the host. They should also better define the importance of heme and heme-containing compounds as iron sources for microbial pathogens within this environment. In addition, these studies should provide insight into the regulatory mechanisms employed by the brucellae to prevent iron toxicity, which may be different from those used by other Gram-negative bacteria that rely on the activity of the ferric uptake regulator (Fur). The proposed studies have public health relevance because they may provide attenuated bacterial strains suitable for testing as novel, live vaccine candidates. Although the Brucella spp. are important zoonotic pathogens and potential bioterrorism agents, there is presently no safe and effective vaccine to prevent human brucellosis, and numerous studies have shown that live, attenuated Brucella strains presently offer the greatest promise for the development of such a vaccine.